• Volume 24, Issue 3

      September 1999,   pages  253-408

    • From fly development to evolution

      J Manjrekar

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    • Neural complexity underlying simple behaviour

      Subhabrata Sanyal Amit Basole

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    • The truth behind the legend: European doctors in pre-colonial India

      Rajesh Kochhar

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      The aim of this article is to point out that the medical history of India in the seventeenth century needs to be studied for its bearing on the history of medical science in this country. During the period 1644–1717, European physicians in India were sought and pampered by the Indian ruling class. English doctors were able to translate this professional goodwill into concrete commercial concessions for the British East India Company. The concessions gave the Company an edge over its rivals, and, more importantly, gave it a cause to fight for. In consequence, the Company was transformed from avaishya (trading) organization into akshatriya (territorial) one. These conclusions warrant a more rigorous professional study of European doctors vis-à-vis their Indian counterparts in the pre-colonial period.

    • Characterization of DNA binding activities of over-expressedKpnI restriction endonuclease and modification methylase

      Siddamadappa Chandrashekaran Padmanabhan Babu Valakunja Nagaraja

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      The genes encoding theKpnI restriction endonuclease and methyltransferase fromKlebsiella pneumoniae have been cloned and expressed inEscherchia coli using a two plasmid strategy. The gene forKpnI methylase with its promoter was cloned and expressed in pACYC184. Even though the methylase clone is in a low copy number plasmid pACMK, high level expression of methylase is achieved. A hyper-expressing clone ofKpnI endonuclease, pETRK was engineered by cloning the R gene into the T7 expression system. This strategy resulted in over-expression ofKpnI endonuclease to about 15–30% of cellular protein. Both the enzymes were purified using a single Chromatographic step to apparent homogeneity. The yield of purified endonuclease and methylase from one liter of culture was approximately 30 and 6 mg respectively. Electrophoretic mobility shift assays show that both the enzymes are capable of binding to specific recognition sequence in the absence of any cofactors. The complexes ofKpnI methyl transferase and endonuclease with their cognate site exhibit distinctive behaviour with respect to ionic requirement.

    • How overproduction of foreign proteins affects physiology of the recombinant strains ofHansenula polymorpha

      Vassili V Velkov Veronika Yu Matys Dmitry M Sokolov

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      Changes in the activity of key enzymes of the methanol utilization pathway of the recombinant strains of methylotrophic yeastHansenula polymorpha R22-2B and LAC-56 were studied at different rates of chemostat growth on methanol containing mineral media. It was shown that the strain R22-2B, initially having a 10-fold increased activity of dihydroxyacetone kinase (DHAK, a key enzyme of formaldehyde assimilation) acquired increased activity of formaldehyde dehydrogenase (FADH, a key enzyme of formaldehyde dissimilation) which resulted in the enhanced oxidation of formaldehyde to CO2. Strain LAC-56, overproducingEscherichia coli β-galactosidase, acquired the decreased intracellular concentration of ATP which resulted in the decrease of the efficiency of formaldehyde assimilation catalyzed by DHAK and resulted in accumulation of toxic formaldehyde. As a consequence some biochemical responses occurred in cells that were directed to a diminishing of the toxic effect of accumulated formaldehyde, namely, the decreasing of methanol oxidase activity (to reduce the rate of formaldehyde synthesis), and the increasing of FADH activity (to increase the rate of formaldehyde oxidation).

    • Cloning and characterization of salicylic acid-induced, intracellular pathogenesis-related gene from tomato (Lycopersicon esculentum)

      C S Sree Vidya M Manoharan G Lakshmi Sita

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      Intracellular pathogenesis-related gene (IPR) from tomato was cloned from a salicylic acid (SA) induced cDNA library and was designated asTSI-1 (tomato stress induced-1). The deduced amino acid sequence ofTSI-1 codes for a 178 amino acid polypeptide of molecular weight 20.4 kDa.TSI-1 is highly homologous to the potatoSTH-2 andSTH-21 IPR and tree pollen allergens which cause type I allergic reactions in humans.TSI-1 lacks a signal peptide like other IPR members. It is organized as a multigene family and is inducible by SA andFusarium oxysporum infection.

    • A bubaline-derived satellite DNA probe uncovers generic affinities of gaur with other bovids

      Pritha Ray Supriya Gangadharan Munmun Chattopadhyay Anu Bashamboo Sunita Bhatnagar Pradeep Kumar Malik Sher Ali

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      DNA typing using genome derived cloned probes may be conducted for ascertaining genetic affinities of closely related species. We analysed gaurBos gaurus, cattleBos indicus, buffaloBubalus bubalis, sheepOvis aries and goatCapra hircus DNA using buffalo derived cloned probe pDS5 carrying an array ofBamHI satellite fraction of 1378 base residues to uncover its genomic organization. Zoo-blot analysis showed that pDS5 does not cross hybridize with non-bovid animals and surprisingly with female gaur genomic DNA. The presence of pDS5 sequences in the gaur males suggests their possible location on the Y chromosome. Genotyping of pDS5 withBamHI enzyme detected mostly monomorphic bands in the bubaline samples and polymorphic ones in cattle and gaur giving rise to clad specific pattern. Similar typing withRsaI enzyme also revealed clad specific band pattern detecting more number of bands in buffalo and fewer in sheep, goat and gaur samples. Copy number variation was found to be prominent in cattle and gaur withRsaI typing. Our data based on matched band profiles (MBP) suggest that gaur is genetically closer to cattle than buffalo contradicting the age-old notion held by some that gaur is a wild buffalo. The pDS5 clone has a potential for estimating the generic and genetic relationship amongst closely related bovid species.

    • Requirement ofwingless signaling andengrailed action in the development and differentiation of reproductive system inDrosophila

      B Starling Emerald J K Roy

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      The segment polarity geneswingless (wg) andengrailed (en) have been shown to play important roles in pattern formation at different stages ofDrosophila development in the thoracic imaginai discs. We have studied the patterns of expression of these genes in genital discs from wild type larvae, pupae and pharate adults and also from hetero-allelic mutant combinations of these genes. Our results suggest that these genes play vital roles in the normal development and differentiation of genital discs and gonads. In the absence of normalwg oren functions, the flies showed a complete lack of internal accessory reproductive organs and specific defects in the external genitalia. In addition, the testes in such males were small, rounded and with an abnormal cellular organization, although the ovaries in females appeared normal. Temperature shift experiments using the conditional mutant allele ofwg, (wgIL-114) indicated a requirement ofwg signaling from second instar onwards for normal development and differentiation of the accessory reproductive organs. Using a heat-shock allele (Hs-wg) we also show that the spatially regulated expression ofwg as a pre-requisite for normal development and differentiation. Based on the expression patterns ofen andhedgehog (hh) we suggest that even in the genital disc development and differentiation the action ofen is mediated throughhh.

    • Phosphorylation and dephosphorylation of Mg2+-independent Ca2+-ATPase from goat spermatozoa

      R Sikdar K Roy A K Mandal P C Sen

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      We reported previously that a Ca2+-ATPase in rat testes and goat spermatozoa could be activated by Ca2+ alone without Mg2+, though it has a lot of similarities with the well known Ca2+, Mg2+-ATPase. Recently, we were successful in isolating the phosphorylated intermediate of the former enzyme under control conditions i.e., in the presence of low concentration of Ca2+ and at low temperature. Increase of the concentration of Ca2+ and/or temperature lead to dephosphorylation. Based on our observations, we proposed a reaction scheme comparable to that of Ca2+, Mg2+-ATPase. The findings strengthened our previous report that Mg2+-independent Ca2+-ATPase is involved in Ca2+ transport and Ca2+ uptake like Ca2+, Mg2+-ATPase.

    • Purification of human seminal acrosin inhibitor and its kinetics

      Aditi Chatterjee Sagarika Kanjilal Asok K Bhattacharyya

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      A low molecular mass, naturally occurring acrosin inhibitor has been identified and purified (490-7-fold) from human semen, and kinetic studies have been performed on the association characteristics as well as for the determination of affinity constants (Ki values). The results show thatKi value (3.34 × 10−2) of the inhibitor towards human acrosin is almost three times lower than that of pancreatic trypsin, indicating a much higher specificity and inhibitory property for acrosin. The purified human seminal acrosin inhibitor has a molecular mass of 5.5 kDa and shows a single band using 10–20% gradient SDS PAGE. The work is of great significance for the development of more specific, nontoxic and irreversible inhibitors for human acrosin.

    • Relationship between power law coefficients and major blood constituents affecting the whole blood viscosity

      Mohammad A Hussain Subir Kar Ram R Puniyani

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      The present work concerns a quantitative analysis of parameters that affect apparent blood viscosity at different low shear rates, i.e, between 1 s−1 and, 100 s−1. Viscosity profile of a large number of blood samples from thromboembolic stroke cases and age and sex matched healthy controls were studied which confirmed non-Newtonian power law behaviour of blood. The power law coefficients,n andk, which are unique to each blood sample, were related with blood viscosity parameters in the form of a mathematical equation by performing non-linear regression analysis. It was possible to calculaten andk of power law model by supplying the values of major blood constituents in the equation obtained for stroke and controls. The calculation ofn andk of a blood sample using the equation obtained, provided a quick information on its apparent viscosity values at any given shear rate without viscometry. The calculated and the experimental viscosity were found in good agreement within a permissible error range. The relation obtained between power law coefficients and major blood constituents in the present investigation would give a quantification of different blood viscosity parameters contributing to the resistance to flow of blood. Such an analysis may be considered as a scientific basis for the study of blood fluidity in different disease conditions.

    • Hydrogen peroxide is a product of oxygen consumption byTrichomonas vaginalis

      Alan Chapman David J Linstead David Lloyd

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      The amitochondriate sexually-transmitted human parasitic protozoanTrichomonas vaginalis (Bushby strain) grown anaerobically on complex medium containing cysteine and ascorbic acid consumed O2 avidly (6.9 μM min−1 per 106 organisms) with an apparentKm value of 5.1 μM O2 : O2 uptake was inhibited by O2 > 120 μM. Spectrophotometric assays in the presence of microperoxidase (419-407 nm) indicated that H2O2 was produced and that inhibition by high O2 concentrations was again evident. Hydrogenosomes oxidizing pyruvate in the presence of ADP and succinate showed similar patterns of O2 consumption, H2O2 production (33.5 pmol min−1 per mg protein), and O2 inhibition. Cytosolic NADH oxidase gave no detectable H2O2, whereas the cytosolic NADPH oxidase produced H2O2 at a rate (43 pmol min−1 per mg protein) greater than that of hydrogenosomes. These results are discussed in relation to the oxidative stress experienced by the pathogen in its natural habitat.

    • Metamorphosis of the central nervous system ofDrosophila melanogaster Meigen (Diptera: Drosophilidae) during pupation

      Kusum Singh R Naresh Singh

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      We studied the metamorphosis of the central nervous system (CNS) and neighbouring muscles ofDrosophila melanogaster (Diptera: Drosophilidae) during pupation by transmission electron microscopy (TEM). The age of white pupa was assumed to be 0 h and the process of metamorphosis was monitored, onward between 6 and 96 h at 25°C. The profiles in the neuropil showed degeneration at 6 h and its extent increased by 12 h. The presence of glycogen in some of these profiles indicated their larval character. Between 12–18 h, the neuronal profiles became separated from one another, the intervening space was filled with extracellular fluid, and some of the larval synapses degenerated. Synaptic vesicles started reappearing around 18 h and synapses were detectable by 24 h. Neuronal processes compactly filled the neuropil by 65 h and the maturation of synapses continued until 86 h. The degeneration of profiles in the neuropil was found to be bimodal, peaking at 12 and 42 h, and that of cortical cells was unimodal with a peak at 42 h. The number of neuronal profiles increased with the development time, indicating that more branching of neuronal profiles occurs in neuropils as the metamorphosis progresses. Average number of synapses per unit area (or volume) is minimum at 18 h and maximum at 72 h, when the average number of synapse per axon profile is 0.54. Because 2 axon profiles share one synapse, a value close to 0.5 for monad synapses shows that, on an average, each axon profile at least makes one synapse at this stage of development. Subsequently, there is more than 75% of reduction in the number of synapses during 73 and 78 h.

      In muscles, vacuoles suggesting histolysis appeared by 6 h. Their ultrastructure became deranged between 12–18 h and myoblasts were found to be present since 8 h. Except for a few muscles in the thorax, such as larval oblique muscles and pharyngeal muscles, most of the muscles in the head and thorax lost all the ultrastructural details and histolyzed by 18 h. Around 38 h, imaginai muscles were detectable, and well-developed muscles were found by 55 h. However, myofibrils continued to be added laterally to the preformed muscles even at 96 h.

      Electron-dense mitochondria (EDMITs) were found in the neuropil, cortex and muscles of pupa, along with mitochondria of characteristic shape and normal appearance. These EDMITs often occurred in large clusters of more than 100, at times near the surface of the tissue. A few of these were enclosed in vacuoles and were darker than the rest of the EDMITs and normal looking mitochondria. Histochemistry with diaminobenzidine showed the presence of cytochrome c and marker enzyme cytochrome oxidase, both in EDMITs and normal mitochondria. EDMITs were not found to be present in any tissue of the adultDrosophila.

    • Electron microscopical studies and restriction analysis ofHelicoverpa armigera nucleo polyhedrosis virus

      S Sudhakar S Mathavan

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      Nuclear polyhedrosis virus ofHelicoverpa armigera (HaNPV) from Madurai (south India) was isolated, purified and subjected to electron microscopical studies. The results indicate that the isolate is a multi embedded form of NPV. Further, STEM observations on polyhedra and polyhedral envelope of the HaMNPV are presented. The restriction pattern and genome size of HaMNPV Madurai isolate are compared with HaNPV isolates reported earlier. A dendrogram is presented to show the relatedness among the isolates.

    • A simple microcapsule generator design for islet encapsulation

      Anandwardhan A Hardikar Makarand V Risbud Ramesh R Bhonde

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      Techniques for immunoisolation and immobilization of viable cells within semipermeable microcapsules have been developed using highly sophisticated droplet generator systems. We propose here an indigenously designed, simple and efficient droplet generator system for encapsulation of the pancreatic islets employing chitosanalginate matrix. The droplet generator system comprises of a needle assembly, a 3-way valve with extended rubber tubing and a filtration unit connected to a pressure pump. Microbeads of the size of around 400 μm diameter or even lesser (minimun attainable size 20.2 μm) could be easily generated using the droplet generator system proposed here. Islet microcapsules cultured in Roswell Park Memorial Institute (RPMI) 1640 with 10% fetal calf serum showed around 98% viability, comparable to that of the non-encapsulated islets. Transplantation of microencapsulated islets to streptozotocin (STZ)-induced diabetic mice, resulted in disappearance of hyperglycemia and restoration of normoglycaemia during a 30-day follow-up suggesting graft functionality. No graft failures were observed in any of the transplanted mice (n = 15) and none of them showed membrane associated fibrous overgrowth, which can be attributed to the fibroblast-growth inhibitory properties of chitosan. The proposed design appears to be superior in its simplicity and cost effectiveness and comparable in performance with the microcapsule generator designs proposed so far. The proposed system can be further exploited for encapsulation and immunoisolation of various cell types in alginate based matrices.

    • Boundaries that demarcate structural and functional domains of chromatin

      Rakesh K Mishra Francois Karch

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      Understanding of how the eukaryotic genome is packaged into chromatin and what the functional consequences of this organization are has begun to emerge recently. The concept of ‘chromatin domains’ — the topologically independent structural unit — is the basis of higher order chromatin organization. The idea that this structural unit may also coincide with the functional unit, offers a useful framework in dissecting the structure-function relationship. Boundaries that define these domains have been identified and several assays have been developed to test themin vivo. We have used genetic means to identify and analyse such boundary elements in the bithorax complex ofDrosophila melanogaster. In this review we discuss chromatin domain boundaries identified in several systems using different means. Although there is no significant sequence conservation among various chromatin domain boundaries, these elements show functional conservation across the species. Finally, we discuss mechanistic aspects of how chromatin domain boundaries may function in organizing and regulating eukaryotic genome.

    • Ascribing functions to the lamin B receptor

      Durgadas P Kasbekar

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      This article reviews the research on the inner nuclear membrane protein lamin B receptor (LBR). It focuses on the biochemical and immunological evidence for an LBR; the cloning of chicken, rat and human LBR cDNAs and genomic sequences; the lamin B-, chromatin-, DNA- and NLS-binding properties of the N-terminal domain and its phosphorylation by different kinases; the sterol C-14 reductase activity of the C-terminal domain; the use of yeast two-hybrid screens and co-immunoprecipitation to identify interacting proteins; and the probing of nuclear assembly and disassembly in living cells with LBR-GFP fusion proteins. The article concludes by considering a scenario whereby LBR levels might even regulate gene expression.

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